IP Library › Granted Patent US 11,634,460

Granted Patent B2

US 11,634,460 · App. 16/754,844 · Granted Apr 25, 2023

Non-neuronal snare-cleaving botulinum neurotoxins

Inventors: Thomas Binz (Wrexham, GB); Stefan Sikorra (Wrexham, GB)

Assignee: IPSEN BIOPHARM LIMITED

C07K14/33 A61K38/4893 A61K38/00

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Quick Facts

Patent No.: US 11,634,460
App. No.: 16/754,844
Granted: Apr 25, 2023
Kind: B2

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Abstract

The present invention provides a modified botulinum neurotoxin A (BoNT/A) L-chain protease that demonstrates enhanced cleaveage of human SNAP-23 (hSNAP-23) relative to unmodified (wild-type) BoNT/A L-chain protease, together with the use thereof for cleaving hSNAP-23.

Claims (67)

1. A modified botulinum neurotoxin A (BoNT/A) L-chain protease comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 1 and

tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1.

2. The modified BoNT/A L-chain protease of claim 1 , comprising alanine at the position corresponding to residue position 29 of SEQ ID NO: 1.

3. The modified BoNT/A L-chain protease of claim 1 , comprising valine at the position corresponding to residue position 166 of SEQ ID NO: 1.

4. The modified BoNT/A L-chain protease of claim 1 , comprising glutamate or aspartate at the position corresponding to residue position 305 of SEQ ID NO: 1.

5. The modified BoNT/A L-chain protease of claim 1 , comprising glutamine, glutamate, or aspartate at the position corresponding to residue position 143 of SEQ ID NO: 1.

6. The modified BoNT/A L-chain protease of claim 1 , comprising glutamate or aspartate at the position corresponding to residue position 251 of SEQ ID NO: 1.

7. A delivery vehicle, comprising:

a) the protease of claim 1 ; and

b) a means for delivering the protease into a target cell.

8. The delivery vehicle of claim 7 , wherein the means for delivering the protease to the target cell comprises:

a) a targeting moiety that binds the delivery vehicle to the target cell; and

b) a translocation peptide that translocates the protease into the target cell.

9. A method of cleaving hSNAP-23, comprising contacting the hSNAP-23 with the protease of claim 1 .

10. The delivery vehicle of claim 7 , comprising a means for delivering the protease into a non-neuronal target cell.

11. The delivery vehicle of claim 8 , wherein the target cell is a non-neuronal target cell.

12. A method of cleaving hSNAP-23, comprising contacting the hSNAP-23 with the protease of claim 7 .

13. The protease of claim 1 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 1.

14. The protease of claim 1 , wherein the amino acid sequence has at least 99% sequence identity to SEQ ID NO: 1.

15. The protease of claim 1 , wherein the amino acid sequence differs from SEQ ID NO: 1 by conservative amino acid substitutions and/or:

(a) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1;

(b) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and lysine at the position corresponding to 312 of SEQ ID NO: 1;

(c) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, phenylalanine at the position corresponding to position 307 of SEQ ID NO: 1, asparagine at the position corresponding to position 308 of SEQ ID NO: 1, and leucine at the position corresponding to 312 of SEQ ID NO: 1;

(d) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, isoleucine at the position corresponding to position 307 of SEQ ID NO: 1, proline at the position corresponding to position 308 of SEQ ID NO: 1, and valine at the position corresponding to 312 of SEQ ID NO: 1;

(e) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, leucine at the position corresponding to position 307 of SEQ ID NO: 1, tyrosine at the position corresponding to position 308 of SEQ ID NO: 1, and methionine at the position corresponding to 312 of SEQ ID NO: 1;

(f) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, leucine at the position corresponding to position 307 of SEQ ID NO: 1, isoleucine at the position corresponding to position 308 of SEQ ID NO: 1, and methionine at the position corresponding to 312 of SEQ ID NO: 1;

(g) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and aspartic acid, glutamic acid, or glutamine at the position corresponding to residue position 143 of SEQ ID NO: 1;

(h) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and aspartic acid at the position corresponding to residue position 305 of SEQ ID NO: 1;

(i) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and alanine at the position corresponding to residue position 29 of SEQ ID NO: 1;

(j) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, alanine at the position corresponding to residue position 29 of SEQ ID NO: 1, and aspartic acid at the position corresponding to residue position 143 of SEQ ID NO: 1;

(k) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, alanine at the position corresponding to residue position 29 of SEQ ID NO: 1, and aspartic acid at the position corresponding to residue position 305 of SEQ ID NO: 1;

(l) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and aspartic acid or glutamic acid at the position corresponding to residue position 251 of SEQ ID NO: 1;

(m) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and aspartic acid at the position corresponding to residue position 256 of SEQ ID NO: 1;

(n) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, alanine at the position corresponding to residue position 29 of SEQ ID NO: 1, aspartic acid at the position corresponding to residue position 143 of SEQ ID NO: 1, and glutamic acid at the position corresponding to residue position 251 of SEQ ID NO: 1;

(o) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, phenylalanine or valine at the position corresponding to residue position 166 of SEQ ID NO: 1, and aspartic acid at the position corresponding to residue position 305 of SEQ ID NO: 1;

(p) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, aspartic acid at the position corresponding to residue position 143 of SEQ ID NO: 1, and phenylalanine at the position corresponding to residue position 166 of SEQ ID NO: 1;

(q) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, alanine at the position corresponding to residue position 29 of SEQ ID NO: 1, phenylalanine or valine at the position corresponding to residue position 166 of SEQ ID NO: 1, and aspartic acid at the position corresponding to residue position 305 of SEQ ID NO: 1;

(r) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, alanine at the position corresponding to residue position 29 of SEQ ID NO: 1, phenylalanine or valine at the position corresponding to residue position 166 of SEQ ID NO: 1, glutamic acid at the position corresponding to residue position 251 of SEQ ID NO: 1, and aspartic acid at the position corresponding to residue position 305 of SEQ ID NO: 1;

(s) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and phenylalanine at residue position 166 of SEQ ID NO: 1;

(t) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, phenylalanine at residue position 166 of SEQ ID NO: 1, and alanine at residue position 254 of SEQ ID NO: 1;

(u) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1, phenylalanine at residue position 166 of SEQ ID NO: 1, alanine at residue position 254 of SEQ ID NO: 1, and aspartic acid at residue position 305 of SEQ ID NO: 1; or

(v) having tyrosine at the position corresponding to residue position 148 of SEQ ID NO: 1 and histidine at residue position 340 of SEQ ID NO: 1.

16. The protease of claim 1 , wherein the amino acid sequence differs from SEQ ID NO: 1 by: